Taxol is a natural compound that possesses impressive anticancer medicinal properties with demonstrated efficacy against carcinomas of the ovary, breast, lung, head and neck, bladder and cervix, melanomas, and AIDS-related Karposi's sarcoma. This outstanding medicinal track record has helped taxol become a very attractive cancer treatment despite formidable manufacturing difficulties. First isolated from the bark of the pacific yew tree, the current production route still depends on isolating a plant-derived taxol intermediate for large-scale manufacture of the final active ingredient by chemical synthesis. Although this semi-synthetic process has eased the toll taken on natural resources, it is still an expensive process that also prevents the synthesis of derivatives with greater potency and a more diverse pharmacological spectrum. These problems can now be addressed through the engineering of microbial cells to produce the drug itself or its key precursor in the semi-synthetic production route, which is the subject of the present application. While microbial synthesis of taxol and its precursors have been actively pursued in recent years, recent advances in metabolic engineering allow a new optimism in addressing this challenge. Specifically, our engineering of the isoprenoid pathway in the bacterium Escherichia coli has led to the increase by more than 100-fold of the production of the first dedicated intermediate in the taxol biosynthetic pathway, taxadiene. Additionally, we have expressed the next gene in the taxol pathway after taxadiene in E. coli. These accomplishments, along with demonstrated expertise of the research team in pathway construction, optimization, and natural product synthesis and functional expression in bacteria and yeasts of genes from plants and other sources that are critical for taxol biosynthesis, support the overall objective of the proposed research, namely, the engineering of microbial metabolism for the efficient synthesis of taxol and its precursors. We will pursue this objective through the following three specific aims: (a) Obtain functional expression of all known genes in the taxol pathway and optimize their activity in conjunction with the upstream isoprenoid pathway for maximum biosynthetic rate;(b) Identify the remaining unknown genes in the taxol pathway (approximately 1/3 of the total) and express them in bacteria and yeast in order to complete the full biosynthetic pathway;(c) Optimize culture conditions and bioreactor operation to maximize taxol production. Our goal, through coordinated pathway and bioreactor engineering, is the development of a scalable microbial fermentation system capable of producing taxol in the gram/liter range. PUBLIC HEALTH RELEVANCE: More efficient production methods would help capitalize on the impressive taxol anticancer properties. In general, it is expected that taxol production would be aided (and, hence, its therapeutic impact expanded) if the biosynthetic pathway could be reconstituted through a simpler heterologous host, one that offered advances in culture growth speed, scalability, and genetic manipulation techniques available to alter and optimize production. Additionally, a heterologous taxol biosynthetic pathway would drastically expand the opportunities of biosynthesizing a vast diversity of taxol derivatives with greater efficacy and broader anticancer properties.